Characteristics of in regeneration of injured peripheral nerves

Ruirui Zhang Nantong University Sailing Chen Nantong University Yinying Shen Nantong University Sheng Yi (  [email protected] ) Nantong University Hui Xu Nantong University

Research

Keywords: peripheral nerve injury, rat sciatic nerve crush injury, sciatic nerve stumps, dorsal root ganglia, upstream cytokines

Posted Date: March 25th, 2020

DOI: https://doi.org/10.21203/rs.3.rs-18898/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Version of Record: A version of this preprint was published on November 23rd, 2020. See the published version at https://doi.org/10.1186/s40779-020-00286-0.

Page 1/16 Abstract

Background

Cytokines are essential cellular modulators of a variety of physiological and pathological activities, including peripheral nerve repair and regeneration. However, the molecular changes of these cellular mediators during peripheral nerve regeneration are not well clarifed. The study is aimed to discover critical cytokines for the regenerative process of injured peripheral nerves.

Methods

The sequencing data of the injured nerve stumps and the dorsal root ganglia (DRGs) of Sprague-Dawley (SD) rats subjected to sciatic nerve (SN) crush injury was analyzed to determine expression patterns of coding for cytokines. PCR experiments were used to validate the accuracy of sequencing data.

Results

A total of 46, 52, and 54 upstream cytokines were differentially expressed in SNs at 1 day, 4 days, and 7 days after nerve injury. And a total of 25, 28, and 34 upstream cytokines were differentially expressed in DRGs at these time points. The expression patterns of some essential upstream cytokines were displayed in a heatmap and validated by PCR experiment. Bioinformatic analysis of these differentially expressed upstream cytokines after nerve injury demonstrated that infammatory and immune responses were signifcantly involved.

Conclusions

In summary, the fndings provided an overview of the dynamic changes of cytokines in SNs and DRGs at different time points after rat nerve crush injury, elucidated the biological processes of differentially expressed cytokines, especially the important roles in infammatory and immune responses during peripheral nerve repair and regeneration, and thus might contribute to identifcation of potential treatments for peripheral nerve repair and regeneration.

1. Introduction

Peripheral nerves are vulnerable tissues that are generally defenseless to traumatic injuries caused by bump, stretch, crush, and penetrating wounds and non-traumatic injuries caused by genetic, metabolic, infectious, and medically induced factors (1, 2). Fortunately, unlike nerves in the central nervous system, peripheral nerves can regenerate and achieve certain functional recovery after injury, although fully functional recovery is generally unexpected (3). After peripheral nerve injury, distal nerve stumps undergo Wallerian degeneration, activated Schwann cells and phagocytosis debris of axon and myelin sheaths, axons of survived neurons regrow toward target tissues for reinnervation (3, 4).

Cytokines are a wide category of immunomodulating or peptides including , , , , and tumour necrosis factors. Cytokines play essential roles in infammation and immune responses and participate in the regulation of the maturation, growth, and responsiveness of a variety of cell populations (5, 6). Cytokines have been identifed to be constitutively involved in the nervous system in health and disease (7–10). Cytokines are also extremely critical for peripheral nerve injury and repair as fne-tuned expressions of cytokines modulate the cellular behaviors of Schwann cells, macrophages, and neurons and regulate debris clearance, axon growth, and peripheral nerve regeneration (11).

Understanding the molecular changes of these cellular mediators during peripheral nerve regeneration opens new possibilities to improve the repair of injured nerves and to minimize the induction of neuropathic pain (11). On this purpose, previously obtained sequencing data of the injured nerve stumps of Sprague-Dawley (SD) rats subjected to sciatic nerve (SN) crush injury was analyzed to determine expression patterns of genes coding for cytokines (12). Moreover, considering that cytokines retrograde transport to the neuronal bodies and affects neuronal activities, sequencing data of the dorsal root ganglia (DRGs) after rat SN crush injury was also jointly investigated (13). Differentially expressed genes in SNs and DRGs after nerve crush injury were identifed and upstream cytokines of these differentially expressed genes were recognized by Ingenuity Pathway Analysis (IPA) bioinformatic tool. Differentially expressed upstream cytokines at 1 day, 4 days, and 7 days after nerve crush injury were subjected

Page 2/16 to functional enrichment of Ontology (GO) categories and Kyoto Enrichment of Genes and Genomes (KEGG) pathways according to Database for Annotation, Visualization, and Integrated Discovery (DAVID).

2. Materials And Methods 2.1. Sequencing data

RNA deep sequencing data of rat SNs at 0 hour, 1 day, 4 days, 7 days, and 14 days after SN crush injury were conserved in National Center for Biotechnology Information (NCBI) database with the accession number PRJNA394957 (SRP113121). Sequencing data of rat DRGs at 0 hour, 3 hours, 9 hours, 1 day, 4 days, and 7 days after SN crush injury were conserved in NCBI database with the accession number PRJNA547681 (SRP200823). Differentially expressed genes in SNs and DRGs at certain time points after nerve crush injury were selected by comparing their expression levels under the injured status with the expression levels under the uninjured status (0 hour control). Genes with a fold changes < 2 or > -2 and a experimental false discovery rate (FDR) < 0.05 were defned as differentially expressed genes. 2.2. Bioinformatic analysis

Upstream cytokines of differentially expressed genes in SNs and DRGs were identifed by IPA bioinformatic tool (Ingenuity Systems Inc., Redwood City, CA, USA) for Ingenuity pathway knowledge base (IPKB)-based upstream regulator analysis. Genes coding for cytokines with a fold changes < 2 or > -2 at 1 day, 4 days, or 7 days as compared with 0 hour were defned as differentially expressed cytokines and were subjected to subsequent bioinformatic analyses.

Commonly differentially expressed cytokines in SNs and DRGs at 1 day, 4 days, or 7 days after SN crush injury were identifed by the Venny 2.1.0 online bioinformatic tool (http://bioinfogp.cnb.csic.es/tools/venny/index.html) (14). The expression profles of these commonly differentially expressed cytokines were demonstrated by a heatmap. Signaling pathways and biological processes involved in differentially expressed upstream cytokines were discovered by DAVID bioinformatic enrichment tools (15, 16). 2.3. Animal surgery and collection of the dorsal root ganglia and SN stumps

The conduction of rat SN crush injury and the collection of SNs and DRGs of uninjured and injured rats were performed as previously described (12, 13). Adult male SD rats weighting 180–220 g were obtained from the Experimental Animal Center of Nantong University (Animal licenses No. SCXK [Su] 2014-0001 and SYXK [Su] 2012-0031) and subjected to animal surgery. Rats were anaesthetizated intraperitoneally with a mixture of 85 mg/kg trichloroacetaldehyde monohydrate, 42 mg/kg magnesium sulfate, and 17 mg/kg sodium pentobarbital. SNs at 10 mm above the bifurcation into the tibial and common fbular nerves were exposed by a skin incision in the left outer mid-thigh. Exposed SNs were crushed with a forceps at a force of 54 N for 3 times with 10 seconds for each time. Rats underwent SN crush injury were sacrifced by decapitation at 1 day, 4 days, and 7 days after animal surgery. Rat underwent sham surgery were sacrifced and designated as 0 hour controls. Rat SNs and lumbar 4 to lumbar 6 DRGs were removed for RNA isolation. 2.4. RNA isolation and PCR validation

RNA was isolated from rat SNs or lumbar 4 to lumbar 6 DRGs using TRIzol reagent (Life Technologies, Carlsbad, CA, USA). Isolated RNA samples were reverse transcribed to cDNA using the Prime-Script reagent kit (TaKaRa, Dalian, Liaoning, China) and subjected to PCR experiments using an Applied Biosystems Stepone System (Applied Biosystems, Foster City, CA, USA) with SYBR Premix Ex Taq (TaKaRa) and specifc primer pairs of target genes (C-X-C motif) 10 (Cxcl10) and 1 receptor antagonist (Il1rn) and reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequences of primer pairs were as follows: Cxcl10 (forward) 5’-GAAGCACCATGAACCCAAGT-3’ and Cxcl10 (reverse) 5’-CAACATGCGGACAGGATAGA-3’; Il1rn (forward) 5’-CTTACCTTCATCCGCTCCGA-3’ and Il1rn (reverse) 5’-GATCAGGCAGTTGGTGGTCAT-3’; and GAPDH (forward) 5’- ACAGCAACAGGGTGGTGGAC-3’ and GAPDH (reverse) 5’-TTTGAGGGTGCAGCGAACTT-3’. Relative mRNA abundances of Cxcl10 −ΔΔCt and Il1rn were determined using the comparative 2 method, in which ΔCt = Ct(injured)-Ct(uninjured) and ΔΔCt = Ct(target gene)-

Ct(reference gene) (17).

Page 3/16 2.5. Statistical analysis

Summarized PCR results were reported as means ± SEM with n = 3. Statistical analysis and graphs were generated using GraphPad Prism 6.0 (GraphPad Software, Inc., San Diego, CA, USA). The p-value was determined from one-way analysis of variance (ANOVA) and a p-value < 0.05 was considered as statistically signifcant.

3. Results 3.1. Identifcation of differentially expressed upstream cytokines in SNs and DRGs following peripheral nerve injury

Previously, the expression patterns of genes in SNs (12) and DRGs (13) at multiple time points after rat SN crush injury were determined and a global view of genetic changes following peripheral nerve injury was obtained. Considering the essential roles of cytokines in tissue remodeling and organ regeneration, IPA bioinformatic analysis was applied to screen upstream cytokines of differentially expressed genes in SNs and DRGs after nerve crush injury. The expression levels of genes coding for these upstream cytokines were further examined and differentially expressed upstream cytokines in SNs and DRGs at 1 day, 4 days, and 7 days after nerve injury were recognized (Table S1).

Page 4/16 Table S1 List of differentially expressed upstream cytokines in SNs and DRGs at 1 day, 4 days, and 7 days after rat SN crush injury.

SN DRG

1d 4d 7d 1d 4d 7d

Gene Log Gene Log Gene Log Gene Log Gene Log Gene Log Ratio Ratio Ratio Ratio Ratio Ratio

Ccl12 14.800 Ccl12 12.409 Ccl12 11.630 Ccl1 7.672 Il5 8.584 Prlh 8.197

Cxcl2 12.446 Cxcl5 9.615 Cxcl2 9.887 Ifna2 6.993 Prlh 8.247 Il24 7.583

Cxcl3 11.164 Cxcl2 9.041 Xcl1 9.833 Il6 6.832 Cd40lg 7.196 Ifna4 7.019

Il10 10.552 Cxcl3 8.656 Cxcl5 9.552 Prl 5.767 Il22 7.147 Il5 6.534

Tnfsf14 9.445 Tnfsf14 8.654 Cxcl3 9.189 Il24 4.839 Ifna4 7.069 Il6 6.316

Il12b 7.123 Il10 8.317 Il17c 8.328 Wnt3a 4.803 Il24 6.951 Ifnb1 6.058

Il1rn 6.515 Il12b 7.491 Il12b 7.287 Il1a 3.991 Il6 6.395 Il9 6.037

Csf2 6.509 Cd40lg 7.118 Il10 7.172 Ctf2 2.669 Il12b 4.247 Prl 5.185

Ccl2 6.353 Ifng 6.695 Tnfsf14 6.772 Cxcl14 1.256 Il1a 3.430 Il22 5.097

Il1b 6.263 Il1a 6.565 Il2 6.317 Ifnk 1.183 Wnt3a 2.857 Csf3 4.824

Il1a 5.728 Csf2 5.361 Ifng 6.228 Tnfsf14 1.084 Ccl22 1.800 Wnt3a 4.391

Ebi3 5.203 Ebi3 5.052 Il1a 5.438 Il11 1.010 Ccl2 1.557 Il1a 3.123

Il36b 5.197 Il1rn 4.870 Wnt3a 5.218 Il12a -1.086 Csf1 1.481 Il12a 1.775

Ifnb1 5.177 Osm 4.488 Il1rn 5.205 Tnfsf10 -1.385 Cxcl14 1.479 Il36rn 1.672

Ifna4 5.139 Il36a 4.383 Il17f 4.911 Il17b -1.501 Cd70 1.459 Ccl22 1.672

Cxcl1 5.127 Ccl2 4.041 Wnt7a 4.643 Ccl19 -1.501 Ccl11 1.450 Csf1 1.528

Il11 4.997 Lif 3.971 Ebi3 4.258 Ccl5 -2.153 Slurp1 1.275 Cd70 1.409

Il6 4.961 Crh 3.921 Tnfsf11 3.650 Cxcl10 -2.365 Lta 1.137 Ccl2 1.202

Osm 4.939 Tnfsf11 3.906 Slurp1 3.650 Tnfsf11 -2.376 Il17c 1.137 Il1b 1.198

Ccl3 4.799 Il36b 3.828 Ccl3 3.494 Cxcl2 -3.724 Tnf 1.000 Il11 1.158

Il2 4.762 Il6 3.658 Il1b 3.375 Wnt1 -3.882 Tnfsf10 -1.060 Cxcl5 1.087

Tnf 4.377 Il1b 3.496 Osm 3.306 Epo -4.641 Crh -1.267 Ctf2 1.087

Prl 4.305 Pf4 3.458 Cd70 2.775 Il17a -4.927 Ccl5 -1.737 Ccl11 1.065

Il17f 3.940 Il18 3.320 Ccl2 2.620 Il10 -5.673 Cxcl10 -3.079 Ifnk 1.035

Wnt3a 3.926 Ccl22 3.222 Lif 2.468 Csf2 -6.302 Epo -4.641 Il1rn 1.010

Lif 3.667 Ccl3 3.158 Pf4 2.463 Csf2 -6.302 Tnfsf10 -1.059

Ccl6 3.458 Cxcl10 3.070 Cxcl10 2.428 Il17b -7.245 Il21 -1.291

Pf4 3.436 Faslg 2.658 Il36b 2.360 Cxcl2 -7.790 Ccl3 -1.372

Cxcl10 3.109 Cd70 2.658 Ccl4 2.241 Tnfsf11 -2.372

Il18 3.093 Tnf 2.643 Tnf 2.109 Cxcl10 -3.337

Timp1 3.087 Il11 2.188 Cxcl14 2.099 Cxcl2 -3.720

Page 5/16 SN DRG

Cd70 2.542 Ccl6 2.173 Il18 2.037 Epo -4.641

Lta 2.307 Ccl5 2.073 Il6 1.945 Il17a -4.927

Tnfsf11 2.027 Cxcl1 2.052 Ccl22 1.891 Csf2 -6.302

Il17a 1.805 Epo 1.880 Ccl28 1.775

Vav3 1.796 Tnfsf13 1.874 Il11 1.775

Ccl22 1.307 Timp1 1.663 Ccl5 1.759

Tnfsf13 1.221 Vav3 1.556 Faslg 1.422

Il33 1.201 Tnfsf13b 1.183 Ccl6 1.412

Aimp1 1.111 Spp1 1.089 Tnfsf13 1.282

Nampt 1.063 Il7 1.073 Vav3 1.204

Il21 -1.121 Wnt4 -1.223 Il17a 1.191

Cntf -1.289 Tnfsf10 -1.490 Scgb1a1 1.191

Tnfsf10 -1.749 Ctf1 -1.844 Il9 1.191

Tnfsf15 -2.474 Il16 -2.032 Dkk3 1.183

Il9 -4.936 Tnfsf15 -2.097 Cxcl1 1.170

Wnt1 -2.249 Fam3b 1.039

Il21 -2.590 Timp1 1.013

Cntf -2.722 Mif -1.046

Il12a -2.802 Il16 -1.171

Il17a -3.913 Tnfsf15 -1.748

Csf3 -5.724 Cntf -2.480

Ccl19 -2.482

Ccl21 -3.616

Venn diagrams were generated to compare differentially expressed upstream cytokines in SNs and DRGs at certain time points after nerve injury and to obtain a comprehensive view of altered cytokines during nerve regeneration (Fig. 1A-1C). A total of 46 upstream cytokines were differentially expressed in SNs at 1 day after nerve injury. At later time points, a relatively larger number of upstream cytokines were differentially expressed in SNs (Fig. 1D). In DRGs, a smaller group of upstream cytokines were differentially expressed as compared with in SNs. And the numbers of differentially expressed upstream cytokines also increased at later time points after nerve injury (Fig. 1D). Detailed investigation of these differentially expressed upstream cytokines showed that the majority of cytokines were up-regulated and only a few cytokines were down-regulated in SNs. However, in DRGs, the percentage of down-regulated cytokines was much higher (Table S1). The intersection set of Venn diagrams discovered cytokines that were differentially expressed in both SNs and DRGs at the same time points. And the expression changes of these intersected cytokines were listed besides the Venn diagrams (Fig. 1A-1C). Some cytokines, such as interleukin-6 (Il6), were kept up-regulated in SNs and DRGs after nerve injury while some cytokines, such as Cxcl10, were up-regulated in SNs but down-regulated in DRGs (Fig. 1A-1C).

3.2. Demonstration of the expression patterns of upstream cytokines in SNs and DRGs following peripheral nerve injury Page 6/16 To identify the dynamic changes of critical cytokines during nerve regeneration, intersected cytokines in SNs and DRGs were further studied. A total of 27 cytokines were differentially expressed in both SNs and DRGs at 1 day, 4 days, or 7 days after nerve injury. The expression levels of these cytokines were investigated and displayed in a heatmap (Fig. 2). Some cytokines showed similar expression treated in both SNs and DRGs. For example, ligand superfamily member 10 (Tnfsf10) was down-regulated in both SNs and DRGs after nerve injury, CD40 ligand (Cd40lg) was up-regulated in both SNs and DRGs at 4 days after nerve injury, and interleukin-9 (Il9) was up-regulated in both SNs and DRGs at 7 days after nerve injury. Some cytokines, such as Il1rn and C-C motif chemokine ligand 2 (Ccl2), exhibited higher expression changes in SNs as compared with DRGs.

The expression patterns of representative cytokines revealed by sequencing assay were further validated by PCR experiments. Different batch of SD rats used for sequencing were collected for sciatic nerve crush injury surgery and subsequent PCR validation. Outcomes from PCR experiments demonstrated that the relative abundances of gene coding for Cxc10 were increased in SNs (Fig. 3A) but decreased in DRGs (Fig. 3B) following nerve injury. And the relative abundances of gene coding for Il1rn were up- regulated in both SNs (Fig. 3C) and DRGs (Fig. 3D). These outcomes were consistent with the expression trends determined by sequencing data (shown in red lines), indicating that sequencing data were of high accuracy. 3.3. Identifcation of signifcantly involved signaling pathways of differentially expressed upstream cytokines following peripheral nerve injury

Bioinformatic analyses were performed to evaluate signifcantly involved signaling pathways of differentially expressed upstream cytokines in SNs and DRGs after nerve injury. Activated signaling pathways that were related to nerve regeneration in up-regulated cytokines and down-regulated cytokines in SNs and DRGs were separately explored (Fig. 4). We focus on upstream cytokines in the current study, so it stands to reason that cytokine-cytokine receptor interaction and chemokine signaling pathway were most strongly enriched signaling pathways. Other signifcantly enriched signaling pathways included Toll-like receptor signaling pathway, TNF signaling pathway, NOD-like receptor signaling pathway, NF-κB signaling pathway, and JAK-STAT signaling pathway. And these signaling pathways were most robustly involved in up-regulated upstream cytokines in SNs.

3.4. Identifcation of signifcantly involved GO biological process categories of differentially expressed upstream cytokines following peripheral nerve injury

Critical nerve regeneration-related biological processes occurred after sciatic nerve crush injury were further discovered by categorizing differentially expressed upstream cytokines to GO terms. Infammatory response and immune response were the most signifcantly involved biological processes and were also most strongly involved in up-regulated upstream cytokines in SNs (Fig. 5). Some other infammatory response and immune response-related biological processes, such as neutrophil chemotaxis, chemotaxis, cellular response to interleukin-1, also exhibited low p-values, indicating the signifcance of infammation and immune response.

To further reveal the importance of infammation in nerve regeneration, infammation-related biological processes were interconnected to a network (Fig. 6A). The infammation-centered network showed that both acute and chronic infammatory responses were activated after nerve repair. The chemotaxis, migration, and extravasation of various types of cells, including , macrophages, and , contribute to activated infammatory response.

Similarly, a network of immune response-related biological processes was also generated (Fig. 6B). Many biological processes related with phenotype modulation of immune cells, such as the activation and proliferation of T cells, B cells, and natural kill cells were signifcantly participated in the generated network, indicating the critical roles of immune cells in nerve repair and regeneration.

4. Discussion

Page 7/16 Peripheral nerve injury induces the disconnection of axons from their cell bodies and leads to the disruption of axons and myelin sheaths in the injured nerve stumps as well as central chromatolysis and nuclear associated changes of somas. With the rapid development of genomics and proteomics, the global genetic and molecular characteristics in a wide variety of physiological and pathological conditions, including peripheral nerve injury and regeneration, were recognized. Many factors were also demonstrated to play fundamental roles in the repair and regeneration of injured peripheral nerves and thus were might be identifed as prospective therapies for the treatment of peripheral nerve injury.

Differentially expressed cytokines in the injured SNs might essentially beneft the infltration and polarization of monocytes, macrophages, and Schwann cells, encourage the phagocytosis and clearance of axon and myelin debris, and promote axon regrowth and regeneration. Actually, a large range of cytokines were found to be up-regulated in the injured nerve stumps. These cytokines might be secreted and released by Schwann cells and macrophages after peripheral nerve injury (18, 19). These up- regulated cytokines, including Ccl2, leukemia inhibitory factor (Lif), tumor necrosis factor-α (Tnf-α), interleukin-1α (Il-1α), interleukin- 1β (I1-1β), and pancreatitis-associated III (Pap-III) recruit the infltration of monocytes and macrophages into injured nerve sites and contribute to the remodeling and reconstruction of the microenvironment surrounding the injured sites (18, 20–23). In our current study, many other cytokines, including chemokine (C-C motif) ligand 12 (Ccl12), C-X-C motif chemokine ligand 2 (Cxcl2), and C-X-C motif chemokine ligand 3 (Cxcl3), were found to be particularly high-expressed in the injured nerve stumps after peripheral nerve injury, indicating the potential applications of these cytokines in treating peripheral nerve injury and promoting axon regeneration.

Moreover, it was worth noting that many cytokines might carry out opposing effects at multiple time points during peripheral nerve regeneration and represent a “double-edged sword” (11). Our current study suggested that differentially expressed upstream cytokines in the injured SNs after peripheral nerve injury were highly related with infammation and immune responses. Therefore, the controversial biological roles of cytokines might be due to the degree and timing of infammation and immune responses induced by different expression levels of cytokines (11). These results were also consistent with our previous fnding that robust immune and infammatory responses were sustained signifcantly involved during nerve degeneration and regeneration (24). These outcomes implied that, to achieve orchestrated regulation of cytokines, it was of great importance to obtain an overview of the expression patterns of cytokines in the injured nerve stumps at different time points after peripheral nerve injury.

Besides affecting the injured nerve stumps and reconstructing the regenerative microenvironment, cytokines could infuence the expressions of neurotrophins and their receptors and thus could affect the neurite outgrowth of neurons (11). For instance, the addition of (IL-4) or -γ (IFN-γ) to neurotrophin-4 (NT-4)-treated DRG neurons would increase NT-4-induced neurite outgrowth and the addition of TNF-α to neurotrophin-treated DRG neurons would decrease neurotrophin-induced neurite outgrowth (25). In addition, cytokine induced infammation and immune response would activate retrograde signaling and might induce the death or survival of DRG neurons (11, 26). Consequently, in our current study, we also jointly determine the dynamic expression levels of cytokines in DRGs and discovered some signifcantly changed cytokines, such as interferon alpha 4 (Ifna4), Il6, and interleukin 24 (Il24).

Interestingly, some cytokines, such as Cxcl10, were discovered to be up-regulated in nerve stumps but down-regulated in DRGs after nerve injury. It was shown that Cxcl10 could promote the invasion of lymphocytes and macrophages and affect myelination in a viral model of multiple sclerosis (27) and could induce neuropathic pain in DRGs after chronic constriction injury (28). Therefore, it was possible that up-regulated Cxcl10 in SNs after nerve injury could contribute to debris clearance in the injured nerve stumps while down-regulated Cxcl10 in DRGs could reduce neuropathic pain. Further functional studies would reveal the specifc roles of these cytokines during peripheral nerve repair and regeneration and would provide new targets of the treatment of peripheral nerve injuries.

5. Conclusions

In summary, the fndings provided an overview of the dynamic changes of cytokines in SNs and DRGs at different time points after rat nerve crush injury, elucidated the biological processes of differentially expressed cytokines, especially the important roles in infammatory and immune responses during peripheral nerve repair and regeneration, and thus might contribute to identifcation of potential treatments for peripheral nerve repair and regeneration.

Page 8/16 Declarations

Authors’ contributions

Conceived and designed the experiments: SY HX. Performed the experiments: RZ XG YS. Analyzed the data: RZ XG HX. Contributed reagents/materials/analysis tools: SY HX. Wrote the manuscript: RZ SY HX.

Acknowledgements

Not applicable.

Funding

This study was supported by the National Natural Science Foundation of China [31700926], Priority Academic Program Development of Jiangsu Higher Education Institutions [PAPD] and Postgraduate Research & Practice Innovation Program of Jiangsu Province [KYCX19_2064].

Availability of data and materials

Availability.

Competing interests

The authors declare that there are no competing interests.

Ethics approval and consent to participate

Animal surgery was ethically approved by the Administration Committee of Experimental Animals, Jiangsu, China and the Institutional Animal Care Guideline of Nantong University and complied with the Guide for the Care and Use of Laboratory Animals approved by the National Institutes of Health.

Consent for publication

Not applicable.

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Figures

Page 10/16 Figure 1

Overview of differentially expressed upstream cytokines in SNs and DRGs after SN crush injury. (A-C) Venn diagram of differentially expressed upstream cytokines in SNs and DRGs at (A) 1 day, (B) 4 days, and (C) 7 days after nerve injury. Overlapped cytokines in SNs and DRGs were listed. (D) The numbers of differentially expressed upstream cytokines were listed.

Page 11/16 Figure 2

Heatmap of the expression levels of commonly differentially expressed upstream cytokines in SNs and DRGs. The relative expression levels of cytokines in SNs and DRGs at 0 hour, 1 day, 4 days, and 7 days were displayed in colors. Green color indicated down-regulation while red color indicated up-regulation. The expression patterns of representative cytokines revealed by sequencing assay were further validated by PCR experiments. Different batch of SD rats used for sequencing were collected for sciatic nerve crush injury surgery and subsequent PCR validation. Outcomes from PCR experiments demonstrated that the relative abundances of gene coding for cytokine Cxc10 were increased in SNs (Figure 3A) but decreased in DRGs (Figure 3B) following nerve injury. And the relative abundances of gene coding for Il1rn were up-regulated in both SNs (Figure 3C) and DRGs (Figure 3D). These outcomes were consistent with the expression trends determined by sequencing data (shown in red lines), indicating that sequencing data were of high accuracy.

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Validation of the expression levels of representative cytokines in SNs and DRGs. (A-B) The relative expression levels of Cxcl10 in (A) SNs and (B) DRGs at 0 hour, 1 day, 4 days, and 7 days after rat SN crush injury. (C-D) The relative expression levels of Il1rn in (C) SNs and (D) DRGs at 0 hour, 1 day, 4 days, and 7 days after rat SN crush injury. The expression levels of Cxcl10 and Il1rn were normalized with GAPDH. Asterisks indicated signifcant differences (p-value<0.05). Red lines indicated the expression trends revealed by sequencing.

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Activated nerve regeneration-related KEGG signaling pathways of differentially expressed upstream cytokines in SNs and DRGs. The sizes of circles indicated the numbers of involved differentially expressed upstream cytokines. Colors indicated the signifcances of KEGG signaling pathways.

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Activated nerve regeneration-related GO biological process categories of differentially expressed upstream cytokines in SNs and DRGs. The sizes of circles indicated the numbers of involved differentially expressed upstream cytokines. Colors indicated the signifcances of GO biological process categories.

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Interaction networks of essential GO biological process categories. (A) Interactions of infammatory response-related GO biological process categories. (B) Interactions of immune response-related GO biological process categories.

Supplementary Files

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SupplementaryTableS1.xlsx

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